Neuromedin B is a neuropeptide originally isolated from spinal cord and belongs to the ranatensin/litorin family of peptides which are related to bombesin. Heterologous antisera against neuromedin B was raised in rabbits and exhibited a wide range of specificities: one antiserum had an absolute specificity for the carboxy terminal tetrapeptide, thus cross-reacting equally well with ranatensin and litorin, but not bombesin; other antisera had slight recognition (0.1-2%) of these peptides. None of the antisera cross-reacted with substance P which shares the carboxy terminal Met-NH2 residue. The appropriate extraction conditions for neuromedin B-related peptides revealed that either acidic or basic conditions yielded the highest concentrations from spinal cord, whereas the incorporation of organic solvents in the extraction method markedly reduced the values obtained.